Micro slide glasses

To examine CCA cell migration, 48-well micro chemotaxis chambers (Neuro Probe, Gaithersburg, MD, USA) were used. Culture medium containing 10% FBS (50 μl) was added to bottom chambers, which were then covered with collagen coated membranes (Neuro Probe, Gaithersburg, MD, USA). For collagen coating, 5 ml of PBS containing 0.02 μg/μl of collagen was treated for 30 min. Cells (5x10⁴) in 50 μl of medium containing 0.05% FBS were seeded in upper chambers. After 5 to 10 hours, cells that migrated through membrane were prepared by fixing and then staining membranes using Diff-quik solution (Sysmex, Kobe, Japan). The stained membranes were washed and were attached on micro slide glasses (Matsunami glass, Osaka, Japan). Experiments were performed in triplicate. Pictures were taken using a light microscope at 40X and 200X. Total numbers of migrated cells were counted using Adobe Photoshop CS6 software. SCR or Mock values were used as controls to calculate relative cell migration.

Daphnia were partially immobilized in minimal amounts of medium on micro slide glasses (Matsunami, Osaka, Japan). Red fluorescence intensity was recorded with a color digital camera (Leica DC500) mounted on a Leica M165C fluorescence microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with a 545 nm excitation and a 620 nm barrier filter. The pictures were taken under 63 × magnification with 100% aperture, 1 s exposure, 3.0 gain, 1.5 saturation, and 1.0 gamma. The red fluorescence intensity of neonates was recorded every 24 h in the first exposure experiment, at day 4 of exposure henceforward.